Humanized antibodies

ABSTRACT

Humanized forms of mouse antibody 3D6 that retain the binding properties of mouse 3D6 are disclosed. Also disclosed are processes for making the humanized antibody, intermediates for making the humanized antibodies, including, nucleotide sequences, vectors, transformed host cells, and methods of using the humanized antibody to treat, prevent, alleviate, reverse, or otherwise ameliorate symptoms or pathology or both, that are associated with Down&#39;s syndrome or pre-clinical or clinical Alzheimer&#39;s disease or cerebral amyloid angiopathy.

This application claims priority of U.S. 60/287,539, filed 2001 Apr. 30,the entire contents of which are incorporated herein by reference.

The invention relates to humanized antibodies useful for treating andpreventing human diseases associated with amyloid β (Aβ), such asAlzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy.Mouse monoclonal antibody 3D6 has been widely used in analyticalmethods. After 3D6 was administered to a group of 11.5-12 month-oldheterozygous, transgenic PDAPP mice (APP^(V717F)) at a weeklyintraperitoneal dose of about 10 mg/kg for six months, it has beenreported that the mice had significantly reduced plaque burden, althoughthe specific location of the reduction was not disclosed. [Bard, F., etal., Nature Med. 6:916-919 (2000); WO 00/72876 and WO 00/72880, 7 Dec.,2000]. It was asserted that the antibody gained access to the centralnervous system in sufficient amounts to “decorate” β-amyloid plaques.Finally, it was stated that mouse 3D6 induces phagocytosis of amyloidplaques in in vitro studies.

Methods for administering aggregated Aβ1-42 to provoke an immunologicresponse and reduced amyloid deposits are described in PCT publicationWO99/27944, published 10 Jun. 1999. The description postulates thatfull-length aggregated Aβ peptide would be a useful immunogen. Theapplication also indicates that antibodies that bind to Aβ peptide couldbe used as alternate therapeutic agents. However, this appears to bespeculation since the supporting data reflect protocols that involveactive immunization using, for example, Aβ1-42.

WO 99/60024, published 25 Nov. 1999, is directed to methods for amyloidremoval using anti-amyloid antibodies. The mechanism, however, is statedto utilize the ability of anti-Aβ antibodies to bind to pre-formedamyloid deposits (i.e. plaques) and result in subsequent microglialclearance of localized plaques. This mechanism was not proved in vivo.This publication further states that to be effective against Aβ plaques,anti-Aβ antibodies must be delivered directly to the brain, becauseantibodies cannot cross the blood brain barrier.

Queen, et al. describe methods of humanizing antibodies [e.g., U.S. Pat.Nos. 5,585,089, 5,693,761, 5,693,762, 6,180,370].

Humanized forms of 3D6 are needed for use in humans having Down'ssyndrome, or pre-clinical or clinical Alzheimer's disease or cerebralamyloid angiopathy (CAA). However, it is not known whether 3D6 can behumanized so that the humanized antibody retained the binding propertiesof the mouse antibody.

SUMMARY OF THE INVENTION

This invention provides humanized forms of 3D6. These humanizedantibodies have binding properties (affinity and epitope location) thatare approximately the same as those of the mouse 3D6 antibody. Theinvention includes antibodies, single chain antibodies, and fragmentsthereof. The invention includes antibodies wherein the CDR are those ofmouse monoclonal antibody 3D6 (sequences SEQ ID NO:1 through SEQ IDNO:6) and wherein the antibodies retain approximately the bindingproperties of the mouse antibody and have in vitro and in vivoproperties functionally equivalent to the mouse antibody. In anotheraspect, this invention provides humanized antibodies and fragmentsthereof, wherein the variable regions have sequences comprising the CDRfrom mouse antibody 3D6 and specific human framework sequences(sequences SEQ ID NO:7-SEQ ID NO:10), wherein the antibodies retainapproximately the binding properties of the mouse antibody and have invitro and in vivo properties functionally equivalent to the mouseantibody 3D6. In another aspect, this invention provides humanizedantibodies and fragments thereof, wherein the light chain is SEQ IDNO:11 and the heavy chain is SEQ ID NO:12.

Also part of the invention are polynucleotide sequences that encode thehumanized antibodies or fragments thereof disclosed above, vectorscomprising the polynucleotide sequences encoding the humanizedantibodies or fragments thereof, host cells transformed with the vectorsor incorporating the polynucleotides that express the humanizedantibodies or fragments thereof, pharmaceutical formulations of thehumanized antibodies and fragments thereof disclosed herein, and methodsof making and using the same.

Such humanized antibodies and fragments thereof are useful for, amongother things, treating and preventing diseases and conditionscharacterized by Aβ plaques or Aβ toxicity in the brain, such asAlzheimer's disease, Down's syndrome, and cerebral amyloid angiopathy inhumans.

The invention also includes use of a humanized antibody of the presentinvention for the manufacture of a medicament, including prolongedexpression of recombinant sequences of the antibody or antibody fragmentin human tissues, for treating, preventing, or reversing Alzheimer'sdisease, Down's syndrome, or cerebral amyloid angiopathy, or to inhibitthe formation of amyloid plaques or the effects of toxic soluble Aβspecies in humans.

DETAILED DESCRIPTION OF THE INVENTION

We have surprisingly found that humanized antibodies, wherein the CDRsoriginate from mouse monoclonal antibody 3D6 and the framework and otherportions of the antibodies originate from a human germ line, bind Aβ1-40and Aβ1-42 with at least the affinity with which mouse 3D6 binds Aβ.Thus, we have a reasonable basis for believing that humanized antibodiesof this specificity, modified to reduce their immunogenicity byconverting them to a humanized form, offer the opportunity to treat,both prophylactically and therapeutically, conditions in humans that areassociated with formation of beta-amyloid plaques. These conditionsinclude, as noted above, pre-clinical and clinical Alzheimer's, Down'ssyndrome, and pre-clinical and clinical cerebral amyloid angiopathy.

As used herein, the word “treat” includes therapeutic treatment, where acondition to be treated is already known to be present andprophylaxis—i.e., prevention of, or amelioration of, the possible futureonset of a condition.

By “antibody” is meant a monoclonal antibody per se, or animmunologically effective fragment thereof, such as an Fab, Fab′, orF(ab′)₂ fragment thereof. In some contexts, herein, fragments will bementioned specifically for emphasis; nevertheless, it will be understoodthat regardless of whether fragments are specified, the term “antibody”includes such fragments as well as single-chain forms. As long as theprotein retains the ability specifically to bind its intended target, itis included within the term “antibody.” Also included within thedefinition “antibody” are single chain forms. Preferably, but notnecessarily, the antibodies useful in the invention are producedrecombinantly. Antibodies may or may not be glycosylated, thoughglycosylated antibodies are preferred. Antibodies are properlycross-linked via disulfide bonds, as is well known.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as kappa and lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 3 or more amino acids.

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. The chainsall exhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarity determining regions or CDRs. The CDRs from the twochains of each pair are aligned by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith well known conventions [Kabat “Sequences of Proteins ofImmunological Interest” National Institutes of Health, Bethesda, Md.,1987 and 1991; Chothia, et al., J. Mol. Biol. 196:901-917 (1987);Chothia, et al., Nature 342:878-883 (1989)].

By “humanized antibody” is meant an antibody that is composed partiallyor fully of amino acid sequences derived from a human antibody germlineby altering the sequence of an antibody having non-human complementaritydetermining regions (CDR). A humanized immunoglobulin does not encompassa chimeric antibody, having a mouse variable region and a human constantregion. However, the variable region of the antibody and even the CDRare humanized by techniques that are by now well known in the art. Theframework regions of the variable regions are substituted by thecorresponding human framework regions leaving the non-human CDRsubstantially intact. As mentioned above, it is sufficient for use inthe methods of the invention, to employ an immunologically specificfragment of the antibody, including fragments representing single chainforms.

Humanized antibodies have at least three potential advantages overnon-human and chimeric antibodies for use in human therapy:

1) because the effector portion is human, it may interact better withthe other parts of the human immune system (e.g., destroy the targetcells more efficiently by complement-dependent cytotoxicity (CDC) orantibody-dependent cellular cytotoxicity (ADCC).

2) The human immune system should not recognize the framework or Cregion of the humanized antibody as foreign, and therefore the antibodyresponse against such an injected antibody should be less than against atotally foreign non-human antibody or a partially foreign chimericantibody.

3) Injected non-human antibodies have been reported to have a half-lifein the human circulation much shorter than the half-life of humanantibodies. Injected humanized antibodies will have a half-lifeessentially identical to naturally occurring human antibodies, allowingsmaller and less frequent doses to be given.

The design of humanized immunoglobulins may be carried out as follows.As to the human framework region, a framework or variable region aminoacid sequence of a CDR-providing non-human immunoglobulin is comparedwith corresponding sequences in a human immunoglobulin variable regionsequence collection, and a sequence having a high percentage ofidentical amino acids is selected. When an amino acid falls under thefollowing category, the framework amino acid of a human immunoglobulinto be used (acceptor immunoglobulin) is replaced by a framework aminoacid from a CDR-providing non-human immunoglobulin (donorimmunoglobulin):

-   -   (a) the amino acid in the human framework region of the acceptor        immunoglobulin is unusual for human immunoglobulin at that        position, whereas the corresponding amino acid in the donor        immunoglobulin is typical for human immunoglobulin at that        position;    -   (b) the position of the amino acid is immediately adjacent to        one of the CDRs; or    -   (c) any side chain atom of a framework amino acid is within        about 5-6 angstroms (center-to-center) of any atom of a CDR        amino acid in a three dimensional immunoglobulin model [Queen,        et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989), and        Co, et al., Proc. Natl. Acad. Sci. USA 88, 2869 (1991)]. When        each of the amino acid in the human framework region of the        acceptor immunoglobulin and a corresponding amino acid in the        donor immunoglobulin is unusual for human immunoglobulin at that        position, such an amino acid is replaced by an amino acid        typical for human immunoglobulin at that position.

A preferred humanized antibody is a humanized form of mouse antibody3D6. The CDRs of humanized 3D6 have the following amino acid sequences:light chain CDR1: 1               5 Lys Ser Ser Gln Ser Leu Leu Asp (SEQID NO:1)  1   10                  15 Ser Asp Gly Lys Thr Tyr Leu Asn

light chain CDR2: 1 5 Leu Val Ser Lys Leu Asp Ser (SEQ ID NO:2)

light chain CDR3: 1 5 Trp Gln Gly Thr His Phe Pro Arg Thr (SEQ ID NO:3)

heavy chain CDR1: 1 5 Asn Tyr Gly Met Ser (SEQ ID NO:4)

heavy chain CDR2: 1               5 Ser Ile Arg Ser Gly Gly Gly Arg Thr(SEQ ID NO:5) 10                  15 Tyr Tyr Ser Asp Asn Val Lys Gly

and, heavy chain CDR3: 1 5 10 Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr.(SEQ ID NO:6)

A preferred light chain variable region of a humanized antibody of thepresent invention has the following amino acid sequence, in which theframework originated from human germline Vk segment DPK19 and J segmentJk4: 1                5 Xaa Val Val Met Thr Gln Xaa Pro (SEQ ID NO:7)   10    10                   15 Leu Xaa Leu Pro Val Thr Xaa Gly             20 Gln Pro Ala Ser Ile Ser Cys Lys  25                   30Ser Ser Gln Ser Leu Leu Asp Ser          35                   40 Asp GlyLys Thr Tyr Leu Asn Trp                    45 Leu Gln Gln Arg Pro GlyGln Ser      50                   55 Pro Xaa Arg Leu Ile Tyr Leu Val              60 Ser Lys Leu Asp Ser Gly Val Pro  65                  70Asp Arg Phe Ser Gly Ser Gly Ser           75                  80 Gly ThrAsp Phe Thr Leu Lys Ile                  85 Ser Arg Val Glu Ala Glu AspGly      90                  95 Val Tyr Tyr Cys Trp Gln Gly            100                 105 Thr His Phe Pro Arg Thr Phe Gly Gly                 110 Gly Thr Lys Xaa Glu Ile Lys Arg

-   -   wherein:    -   Xaa at position 1 is Asp or Tyr;    -   Xaa at position 7 is Ser or Thr;    -   Xaa at position 10 is Ser or Thr;    -   Xaa at position 15 is Leu, Ile, or Val;    -   Xaa at position 50 is Arg or Lys;    -   Xaa at position 88 is Val or Leu; and    -   Xaa at position 109 is Val or Leu.

A preferred heavy chain variable region of a humanized antibody of thepresent invention has the following amino acid sequence, in which theframework originated from human germline VH segment DP-45 and J segmentJH4, with several amino acid substitutions to the consensus amino acidsin the same human subgroup to reduce potential immunogenicity: 1 5 10 15Glu Val Xaa Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly (SEQ IDNO:8) 20 25 30 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe SerAsn Tyr 35 40 45 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val 50 55 60 Ala Ser Ile Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser AspAsn Val 65 70 75 80 Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys AsnXaa Leu Tyr 85 90 95 Leu Gln Met Asn Ser Leu Xaa Xaa Glu Asp Thr Ala ValTyr Tyr Cys 100 105 110 Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp TyrTrp Gly Gln Gly 115 Thr Xaa Val Thr Val Ser Ser

-   -   wherein:    -   Xaa at position 3 is Gln, Lys, or Arg;    -   Xaa at position 78 is Ser or Thr;    -   Xaa at position 87 is Arg or Lys;    -   Xaa at position 88 is Ala, Ser, or Thr; and    -   Xaa at position 114 is Leu, Thr, Ile, or Val.

A particularly preferred light chain variable region of a humanizedantibody of the present invention has the following amino acid sequence,in which the framework originated from human germline Vk segment DPK19and J segment Jk4: 1 5 10 15 Asp Val Val Met Thr Gln Ser Pro Leu Ser LeuPro Val Thr Leu Gly (SEQ ID NO:9) 20 25 30 Gln Pro Ala Ser Ile Ser CysLys Ser Ser Gln Ser Leu Leu Asp Ser 35 40 45 Asp Gly Lys Thr Tyr Leu AsnTrp Leu Gln Gln Arg Pro Gly Gln Ser 50 55 60 Pro Arg Arg Leu Ile Tyr LeuVal Ser Lys Leu Asp Ser Gly Val Pro 65 70 75 80 Asp Arg Phe Ser Gly SerGly Ser Gly Thr Asp Phe Thr Leu Lys Ile 85 90 95 Ser Arg Val Glu Ala GluAsp Val Gly Val Tyr Tyr Cys Trp Gln Gly 100 105 110 Thr His Phe Pro ArgThr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg.

A particularly preferred heavy chain variable region of a humanizedantibody of the present invention has the following amino acid sequence,in which the framework originated from human germline VH segment DP-45and J segment JH4: 1 5 10 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal Gln Pro Gly Gly (SEQ ID NO:10) 20 25 30 Ser Leu Arg Leu Ser Cys AlaGly Ser Gly Phe Thr Phe Ser Asn Tyr 35 40 45 Gly Met Ser Trp Val Arg GlnAla Pro Gly Lys Gly Leu Glu Trp Val 50 55 60 Ala Ser Ile Arg Ser Gly GlyGly Arg Thr Tyr Tyr Ser Asp Asn Val 65 70 75 80 Lys Gly Arg Phe Thr IleSer Arg Glu Asn Ala Lys Asn Ser Leu Tyr 85 90 95 Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 100 105 110 Val Arg Tyr Asp HisTyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 115 Thr Leu Val Thr Val SerSer.

A preferred light chain for a humanized antibody of the presentinvention has the amino acid sequence: 1 5 10 15 Asp Val Val Met Thr GlnSer Pro Leu Ser Leu Pro Val Thr Leu Gly (SEQ ID NO:11} 20 25 30 Gln ProAla Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 35 40 45 Asp GlyLys Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Ser 50 55 60 Pro ArgArg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 65 70 75 80 AspArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 85 90 95 SerArg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 100 105 110Thr His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 115 120135 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn AlaLeu Gln 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp SerLys Asp Ser 180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser LysAla Asp Tyr Glu 195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr HisGln Gly Leu Ser Ser 210 215 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys.

A preferred heavy chain for a humanized antibody of the presentinvention has the amino acid sequence: 1 5 10 15 Gln Val Gln Leu Val GlnSer Gly Gly Gly Leu Val Gln Pro Gly Gly (SEQ ID NO:12) 20 25 30 Ser LeuArg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Asn Tyr 35 40 45 Gly MetSer Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 50 55 60 Ala SerIle Arg Ser Gly Gly Gly Arg Thr Tyr Tyr Ser Asp Asn Val 65 70 75 80 LysGly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Ser Leu Tyr 85 90 95 LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 100 105 110Val Arg Tyr Asp His Tyr Ser Gly Ser Ser Asp Tyr Trp Gly Gln Gly 115 120125 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 130135 140 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu145 150 155 160 Gly Cys Leu Val Lys Asp Tyr Phe Pro Gln Pro Val Thr ValSer Trp 165 170 175 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProAla Val Leu 180 185 190 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val ValThr Val Pro Ser 195 200 205 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys AsnVal Asn His Lys Pro 210 215 220 Ser Asn Thr Lys Val Asp Lys Lys Val GluPro Lys Ser Cys Asp Lys 225 230 235 240 Thr His Thr Cys Pro Pro Cys ProAla Pro Gln Leu Leu Gly Gly Pro 245 250 255 Ser Val Phe Leu Phe Pro ProLys Pro Lys Asp Thr Leu Met Ile Ser 260 265 270 Arg Thr Pro Glu Val ThrCys Val Val Val Asp Val Ser His Glu Asp 275 280 285 Pro Glu Val Lys PheAsn Trp Tyr Val Asp Gly Val Glu Val His Asn 290 295 300 Ala Lys Thr LysPro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 305 310 315 320 Val SerVal Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 325 330 335 TyrLys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 340 345 350Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 355 360365 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 370375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrVal Asp Lys 420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys SerVal Met His Glu 435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser LeuSer Leu Ser Pro Gly Lys

Other sequences are possible for the light and heavy chains forhumanized 3D6. The immunoglobulins can have two pairs of lightchain/heavy chain complexes, at least one chain comprising one or moremouse complementarity determining regions functionally joined to humanframework region segments.

In another aspect, the present invention is directed to recombinantpolynucleotides encoding antibodies which, when expressed, comprise theheavy and light chain CDRs from an antibody of the present invention.Exemplary polynucleotides, which on expression code for the polypeptidechains comprising the heavy and light chain CDRs of monoclonal antibody3D6 are given herein. Due to codon degeneracy, other polynucleotidesequences can be readily substituted for those sequences. Particularlypreferred polynucleotides of the present invention encode antibodies,which when expressed, comprise the CDRs of SEQ ID NO:1-SEQ ID NO:6, orany of the variable regions of SEQ ID NO:7-SEQ ID NO:10, or the lightand heavy chains of SEQ ID NO:11 and SEQ ID NO:12.

The polynucleotides will typically further include an expression controlpolynucleotide sequence operably linked to the humanized immunoglobulincoding sequences, including naturally-associated or heterologouspromoter regions. Preferably, the expression control sequences will beeukaryotic promoter systems in vectors capable of transforming ortransfecting eukaryotic host cells, but control sequences forprokaryotic hosts may also be used. Once the vector has beenincorporated into the appropriate host cell line, the host cell ispropagated under conditions suitable for expressing the nucleotidesequences, and, as desired, the collection and purification of the lightchains, heavy chains, light/heavy chain dimers or intact antibodies,binding fragments or other immunoglobulin forms may follow.

The nucleic acid sequences of the present invention capable ofultimately expressing the desired humanized antibodies can be formedfrom a variety of different polynucleotides (genomic or cDNA, RNA,synthetic oligonucleotides, etc.) and components (e.g., V, J, D, and Cregions), using any of a variety of well known techniques. Joiningappropriate genomic and synthetic sequences is a common method ofproduction, but cDNA sequences may also be utilized.

Below is a cDNA sequence (SEQ ID NO:17), from which the light chainhaving the amino acid sequence of SEQ ID NO:19 may be expressed.ATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGT  1---------+---------+---------+---------+---------+---------+  60M  M  S  P  A  Q  F  L  F  L  L  V  L  W  I  R  E  T  N  GGATGTTGTGATGACCCAGTCTCCACTCTCCTTGCCTGTTACCCTGGGACAACCAGCCTCC  61---------+---------+---------+---------+---------+---------+ 120D  V  V  M  T  Q  S  P  L  S  L  P  V  T  L  G  Q  P  A  SATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGG 121---------+---------+---------+---------+---------+---------+ 180I  S  C  K  S  S  Q  S  L  L  D  S  D  G  K  T  Y  L  N  WTTGCAACAGCGCCCAGGCCAGTCTCCAAGACGCCTAATCTATCTGGTGTCTAAACTGGAC 181---------+---------+---------+---------+---------+---------+ 240L  Q  Q  R  P  G  Q  S  P  R  R  L  I  Y  L  V  S  K  L  DTCTGGAGTCCCTGACAGGTTCTCTGGCAGTGGATCAGGGACAGATTTTACACTGAAAATC 241---------+---------+---------+---------+---------+---------+ 300S  G  V  P  D  R  F  S  G  S  G  S  G  T  D  F  T  L  K  IAGCAGAGTCGAGGCTGAGGATGTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCT 301---------+---------+---------+---------+---------+---------+ 360S  R  V  E  A  E  D  V  G  V  Y  Y  C  W  Q  G  T  H  F  PCGGACGTTCGGTGGAGGCACCAAGGTGGAAATCAAACGTACTGTGGCTGCACCATCTGTC 361---------+---------+---------+---------+---------+---------+ 420R  T  F  G  G  G  T  K  V  E  I  K  R  T  V  A  A  P  S  VTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTG 421---------+---------+---------+---------+---------+---------+ 480F  I  F  P  P  S  D  E  Q  L  K  S  G  T  A  S  V  V  C  LTCGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAA 481---------+---------+---------+---------+---------+---------+ 540L  N  N  F  Y  P  R  E  A  K  V  Q  W  K  V  D  N  A  L  QTCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTC 541---------+---------+---------+---------+---------+---------+ 600S  G  N  S  Q  E  S  V  T  E  Q  D  S  K  D  S  T  Y  S  LAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA 601---------+---------+---------+---------+---------+---------+ 660S  S  T  L  T  L  S  K  A  D  Y  E  K  H  K  V  Y  A  C  EGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO:17)661 ---------+---------+---------+---------+---------+---------+ 720V  T  H  Q  G  L  S  S  P  V  T  K  S  F  N  R  G  E  C (SEQ ID NO:19)

Below is a cDNA sequence (SEQ ID NO:18), from which the heavy chainhaving the amino acid sequence of SEQ ID NO:20 may be expressed.ATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTCTTAAAAGGTGTCCAGTGTGAA 1---------+---------+---------+---------+---------+---------+  60M  N  F  G  L  S  L  I  F  L  V  L  V  L  K  G  V  Q  C  EGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGCTCTCTGAGGCTCTCC 61---------+---------+---------+---------+---------+---------+  120V  Q  L  V  E  S  G  G  G  L  V  Q  P  G  G  S  L  R  L  STGTGCAGGCTCTGGATTCACTTTCAGTAACTATGGCATGTCTTGGGTTCGCCAGGCTCCT 121---------+---------+---------+---------+---------+---------+  180C  A  G  S  G  F  T  F  S  N  Y  G  M  S  W  V  R  Q  A  PGGAAAGGGACTGGAGTGGGTTGCATCCATTAGGAGTGGTGGTGGTAGAACCTACTATTCA 181---------+---------+---------+---------+---------+---------+  240G  K  G  L  E  W  V  A  S  I  R  S  G  G  G  R  T  Y  Y  SGACAATGTAAAGGGCCGATTCACCATCTCCAGAGAGAATGCCAAGAACAGCCTGTACCTG 241---------+---------+---------+---------+---------+---------+  300D  N  V  K  G  R  F  T  I  S  R  E  N  A  K  N  S  L  Y  LCAAATGAACAGTCTGAGAGCTGAGGACACGGCTGTCTATTATTGTGTCAGATATGATCAC 301---------+---------+---------+---------+---------+---------+  360Q  M  N  S  L  R  A  E  D  T  A  V  Y  Y  C  V  R  Y  D  HTATAGTGGTAGCTCCGACTACTGGGGCCAGGGCACCTTGGTCACAGTCTCCTCAGCCTCC 361---------+---------+---------+---------+---------+---------+  420Y  S  G  S  S  D  Y  W  G  Q  G  T  L  V  T  V  S  S  A  SACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACA 421---------+---------+---------+---------+---------+---------+  480T  K  G  P  S  V  F  P  L  A  P  S  S  K  S  T  S  G  G  TGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAAC 481---------+---------+---------+---------+---------+---------+  540A  A  L  G  C  L  V  K  D  Y  F  P  E  P  V  T  V  S  W  NTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTC 541---------+---------+---------+---------+---------+---------+  600S  G  A  L  T  S  G  V  H  T  F  P  A  V  L  Q  S  S  G  LTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATC 601---------+---------+---------+---------+---------+---------+  660Y  S  L  S  S  V  V  T  V  P  S  S  S  L  G  T  Q  T  Y  ITGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCT 661---------+---------+---------+---------+---------+---------+  720C  N  V  N  H  K  P  S  N  T  K  V  D  K  K  V  E  P  K  STGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCA 721---------+---------+---------+---------+---------+---------+  780C  D  K  T  H  T  C  P  P  C  P  A  P  E  L  L  G  G  P  SGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTC 781---------+---------+---------+---------+---------+---------+  840V  F  L  F  P  P  K  P  K  D  T  L  M  I  S  R  T  P  E  VACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTG 841---------+---------+---------+---------+---------+---------+  900T  C  V  V  V  D  V  S  H  E  D  P  E  V  K  F  N  W  Y  VGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACG 901---------+---------+---------+---------+---------+---------+  960D  G  V  E  V  H  N  A  K  T  K  P  R  E  E  Q  Y  N  S  TTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTAC 961---------+---------+---------+---------+---------+---------+ 1020Y  R  V  V  S  V  L  T  V  L  H  Q  D  W  L  N  G  K  E  YAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCC 1021---------+---------+---------+---------+---------+---------+ 1080K  C  K  V  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  AAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACC 1081---------+---------+---------+---------+---------+---------+ 1140K  G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  D  E  L  TAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG 1141---------+---------+---------+---------+---------+---------+ 1200K  N  Q  V  S  L  T  C  L  V  K  G  F  Y  P  S  D  I  A  VGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC 1201---------+---------+---------+---------+---------+---------+ 1260E  W  E  S  N  G  Q  P  E  N  N  Y  K  T  T  P  P  V  L  DTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAG 1261---------+---------+---------+---------+---------+---------+ 1320S  D  G  S  F  F  L  Y  S  K  L  T  V  D  K  S  R  W  Q  QGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG 1321---------+---------+---------+---------+---------+---------+ 1380G  N  V  F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  KAGCCTCTCCCTGTCTCCGGGTAAA (SEQ ID NO:18) 1381---------+---------+---------+------ 1416 S  L  S  L  S  P  G  K (SEQ IDNO:20)

The complete sequence of a humanized 3D6 light chain gene with introns(located between MluI and BamHI sites, as in pVk-Hu3D6) is shown below(SEQ ID NO:15). The nucleotide number indicates its position inpVk-Hu3D6. The Vk and Ck exons are translated in single letter code; thedot indicates the translation termination codon. The mature light chainstarts at the double-underlined aspartic acid (D). The intron sequenceis in italics. The polyA signal is underlined. The expressed light chaincorresponds to SEQ ID NO:11 when mature. 619ACGCGTCCACCATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGTTGTG            M  M  S  P  A  Q  F  L  F  L  L  V  L  W  I  R  E  T  N  G  D   V  V 699ATGACCCAGTCTCCACTCTCCTTGCCTGTTACCCTGGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTT M  T  Q  S  P  L  S  L  P  V  T  L  G  Q  P  A  S  I  S  C  K  S  S  Q  S  L  L779AGATAGTGATGGAAAGACATATTTGAATTGGTTGCAACAGCGCCCAGGCCAGTCTCCAAGACGCCTAATCTATCTGGTGT  D  S  D  G  K  T  Y  L  N  W  L  Q  Q  R  P  G  Q  S  P  R  R  L  I  Y  L  V859CTAAACTGGACTCTGGAGTCCCTGACAGGTTCTCTGGCAGTGGATCAGGGACAGATTTTACACTGAAAATCAGCAGAGTCS  K  L  D  S  G  V  P  D  R  F  S  G  S  G  S  G  T  D  F  T  L  K  I  S  R  V939GAGGCTGAGGATGTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCGGACGTTCGGTGGAGGCACCAAGGTGGA E  A  E  D  G  V  Y  Y  C  W  Q  G  T  H  F  P  R  T  F  G  G  G  T  K  V  E1019AATCAAACGTAAGTGCACTTTCCTTCTAGAATTCTAAACTCTGAGGGGGTCGGATGACGTGGCCAATTCTTTGCCTAAAG  I  K  R 1099CATTGAGTTTACTGCAAGGTCAGAAAAGCATGCAAAGCCCTCAGAATGGCTGCAAAGAGCTCCAACAAAACAATTTAGAA1179CTTTATTAAGGAATAGGGGGAAGCTAGGAAGAAACTCAAAACATCAAGATTTTAAATACGCTTCTTGGTCTCCTTGCTAT1259AATTATCTGGGATAAGCATGCTGTTTTCTGTCTGTCCCTAACATGCCCTGTGATTATCCGCAAACAACACACCCAAGGGC1339AGAACTTTGTTACTTAAACACCATCCTGTTTGCTTCTTTCCTCAGGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCG                                                T  V  A  A  P  S  V  F  I  F  P1419CCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAAGT P  S  D  E  Q  L  K  S  G  T  A  S  V  V  C  L  L  N  N  F  Y  P  R  E  A  K  V1499ACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTGCACAGAGCAGGACAGCAAGGACAGCACCT  Q  W  K  V  D  N  A  L  Q  S  G  N  S  Q  E  S  V  T  E  Q  D  S  K  D  S  T1579ACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGY  S  L  S  S  T  L  T  L  S  K  A  D  Y  E  K  H  K  V  Y  A  C  E  V  T  H  Q1659GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAGGGAGAAGTGCCCCCACCTGCTCCTCAGTTC G  L  S  S  P  V  T  K  S  F  N  R  G  E  C  ● 1739CAGCCTGACCCCCTCCCATCCTTTGGCCTCTGACCCTTTTTCCACAGGGGACCTACCCCTATTGCGGTCCTCCAGCTCAT1819CTTTCACCTCACCCCCCTCCTCCTCCTTGGCTTTAATTATGCTAATGTTGGAGGAGAATGAATAAATAAA  GTGAATCTTT1899GCACCTGTGGTTTCTCTCTTTCCTCATTTAATAATTATTATCTGTTGTTTTACCAACTACTCAATTTCTCTTATAAGGGA1979CTAAATATGTAGTCATCCTAAGGCGCATAACCATTTATAAAATCATCCTTCATTCTATTTTACCCTATCATCCTCTGCA2059AGACAGTCCTCCCTCAAACCCACAAGCCTTCTGTCCTCACAGTCCCCTGGGCCATGGTAGGAGAGACTTGCTTCCTTGTT2139TTCCCCTCCTCAGCAAGCCCTCATAGTCCTTTTTAAGGGTGACAGGTCTTACAGTCATATATCCTTTGATTCAATTCCCT2219GAGAATCAACCAAAGCAAATTTTTCAAAAGAAGAAACCTGCTATAAAGAGAATCATTCATTGCAACATGATATAAAATAA2299CAACACAATAAAAGCAATTAAATAAACAAACAATAGGGAAATGTTTAAGTTCATCATGGTACTTAGACTTAATGGAATGT2379CATGCCTTATTTACATTTTTAAACAGGTACTGAGGGACTCCTGTCTGCCAAGGGCCGTATTGAGTACTTTCCACAACCTA2459ATTTAATCCACACTATACTGTGAGATTAAAAACATTCATTAAAATGTTGCAAAGGTTCTATAAAGCTGAGAGACAAATAT2539 ATTCTATAACTCAGCAATCCCACTTCTAGGATC (SEQ ID NO:15)

The complete sequence of a humanized 3D6 heavy chain gene with introns(located between MluI and BamHI sites, as in pVg1-Hu3D6) is shown below(SEQ ID NO:16). The nucleotide number indicates its position inpVg1-Hu3D6. The VH and CH exons are translated in single letter code;the dot indicates the translation termination codon. The mature heavychain starts at the double-underlined glutamine (Q). The intronsequences are in italic. The polyA signal is underlined. The expressedheavy chain corresponds to SEQ ID NO:12 when mature. 619ACGCGTCCACCATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTCTTAAAAGGTGTCCAGTGTGAAGTGCAACTG            M  N  F  G  L  S  L  I  F  L  V  L  V  L  K  G  V  Q  C  E   V  Q  L 699GTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGCTCTCTGAGGCTCTCCTGTGCAGGCTCTGGATTCACTTTCAGTAA V  E  S  G  G  G  L  V  Q  P  G  G  S  L  R  L  S  C  A  G  S  G  F  T  F  S  N779CTATGGCATGTCTTGGGTTCGCCAGGCTCCTGGAAAGGGACTGGAGTGGGTTGCATCCATTAGGAGTGGTGGTGGTAGAA  Y  G  M  S  W  V  R  Q  A  P  G  K  G  L  E  W  V  A  S  I  R  S  G  G  G  R859CCTACTATTCAGACAATGTAAAGGGCCGATTCACCATCTCCAGAGAGAATGCCAAGAACAGCCTGTACCTGCAAATGAACT  Y  Y  S  D  N  V  K  G  R  F  T  I  S  R  E  N  A  K  N  S  L  Y  L  Q  M  N939AGTCTGAGAGCTGAGGACACGGCTGTCTATTATTGTGTCAGATATGATCACTATAGTGGTAGCTCCGACTACTGGGGCCA S  L  R  A  E  D  T  A  V  Y  Y  C  V  R  Y  D  H  Y  S  G  S  S  D  Y  W  G  Q1019GGGCACCTTGGTCACAGTCTCCTCAGGTGAGTCCTCACAACCTCTAGAGCTTTCTGGGGCAGGCCAGGCCTGACCTTGGC  G  T  L  V  T  V  S  S 1099TTTGGGGCAGGGAGGGGGCTAAGGTGAGGCAGGTGGCGCCAGCCAGGTGCACACCCAATGCCCATGAGCCCAGACACTGG1179ACGCTGAACCTCGCGGACAGTTAAGAACCCAGGGGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCACACCGCGGTCACATG1259GCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGC                  A  S  T  K  G  P  S  V  F  P  L  A  P  S  S  K  S  T  S  G  G1339ACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAG T  A  A  L  G  C  L  V  K  D  Y  F  P  E  P  V  T  V  S  W  N  S  G  A  L  T  S1419CGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCA  G  V  H  T  F  P  A  V  L  Q  S  S  G  L  Y  S  L  S  S  V  V  T  V  P  S  S1499GCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGGTGAGAGGS  L  G  T  Q  T  Y  I  C  N  V  N  H  K  P  S  N  T  K  V  D  K  K  V1579CCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAG1659TCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTC1739TTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACACAAAGGGGCAGGTGC1819TGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCAC1899TCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTGACE  P  K  S  C  D 1979AAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAG K  T  H  T  C  P  P  C  P 2059AGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGA  P  E  L  L 2139GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGT G  G  P  S  V  F  L  F  P  P  K  P  K  D  T  L  M  I  S  R  T  P  E  V  T  C  V2219GGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA  V  V  D  V  S  H  E  D  P  E  V  K  F  N  W  Y  V  D  G  V  E  V  H  N  A  K2299CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATT  K  P  R  E  E  Q  Y  N  S  T  Y  R  V  V  S  V  L  T  V  L  H  Q  D  W  L  N2379GGCAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGG G  K  E  Y  K  C  K  V  S  N  K  A  L  P  A  P  I  E  K  T  I  S  K  A  K2459GACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTTACCAACC2539TCTGTCCCTACAGGGCAGCCCCTGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGT             G  Q  P  R  E  P  Q  V  Y  T  L  P  P  S  R  D  E  L  T  K  N  Q  V2619CAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAATGGGCAGCCGGAGAACA  S  L  T  C  L  V  K  G  F  Y  P  S  D  I  A  V  E  W  E  S  N  G  Q  P  E  N2699ACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGN  Y  K  T  T  P  P  V  L  D  S  D  G  S  F  F  L  Y  S  K  L  T  V  D  K  S  R2779TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCT W  Q  Q  G  N  V  F  S  C  S  V  M  H  E  A  L  H  N  H  Y  T  Q  K  S  L  S  L2859GTCTCCGGGTAAATGAGTGCGACGGCCGGCAAGCCCCCGCTCCCCGGGCTCTCGCGGTCGCACGAGGATGCTTGGCACGT  S  P  G  K  ● 2939ACCCCCTGTACATACTTCCCGGGCGCCCAGCATGGAAATAAA GCACCCAGCGCTGCCCTGGGCCCCTGCGAGACTGTGAT3019GGTTCTTTCCACGGGTCAGGCCGAGTCTGAGGCCTGAGTGGCATGAGGGAGGCAGAGCGGGTCCCACTGTCCCCACACTG3099GCCCAGGCTGTGCAGGTGTGCCTGGGCCGCCTAGGGTGGGGCTCAGCCAGGGGCTGCCCTCGGCAGGGTGGGGGATTTGC3179CAGCGTGGCCCTCCCTCCAGCAGCACCTGCCCTGGGCTGGGCCACGGGAAGCCCTAGGAGCCCCTGGGGACAGACACACA3259GCCCCTGCCTCTGTAGGAGACTGTCCTGTTCTGTGAGCGCCCTGTCCTCCGACCTCCATGCCCACTCGGGGGCATGCCTA3339GTCCATGTGCGTAGGGACAGGCCCTCCCTCACCCATCTACCCCCACGGCACTAACCCCTGGCTGCCCTGCCCAGCCTCGC3419ACCCGCATGGGGACACAACCGACTCCGGGGACATGCACTCTCGGGCCCTGTGGAGGGACTGGTGCAGATGCCCACACACA3499CACTCAGCCCAGACCCGTTCAACAAACCCCGCACTGAGGTTGGCCGGCCACACGGCCACCACACACACACGTGCACGCCT3579CACACACGGAGCCTCACCCGGGCGAACTGCACAGCACCCAGACCAGAGCAAGGTCCTCGCACACGTGAACACTCCTCGGA3659CACAGGCCCCCACGAGCCCCACGCGGCACCTCAAGGCCCACGAGCCTCTCGGCAGCTTCTCCACATGCTGACCTGCTCAG3739ACAAACCCAGCCCTCCTCTCACAAGGGTGCCCCTGCAGCCGCCACACACACACAGGGGATCACACACCACGTCACGTCCC3819 TGGCCCTGGCCCACTTCCCAGTGCCGCCCTTCCCTGCAGGATCC (SEQ ID NO:16)

Human constant region DNA sequences can be isolated in accordance withwell known procedures from a variety of human cells, but preferably fromimmortalized B-cells. Suitable source cells for the polynucleotidesequences and host cells for immunoglobulin expression and secretion canbe obtained from a number of sources well-known in the art.

In addition to the humanized immunoglobulins specifically describedherein, other “substantially homologous” modified immunoglobulins can bereadily designed and manufactured utilizing various recombinant DNAtechniques well known to those skilled in the art. For example, theframework regions can vary from the native sequences at the primarystructure level by several amino acid substitutions, terminal andintermediate additions and deletions, and the like. Moreover, a varietyof different human framework regions may be used singly or incombination as a basis for the humanized immunoglobulins of the presentinvention. In general, modifications of the genes may be readilyaccomplished by a variety of well-known techniques, such assite-directed mutagenesis.

Alternatively, polypeptide fragments comprising only a portion of theprimary antibody structure may be produced, which fragments possess oneor more immunoglobulin activities (e.g., complement fixation activity).These polypeptide fragments may be produced by proteolytic cleavage ofintact antibodies by methods well known in the art, or by inserting stopcodons at the desired locations in vectors using site-directedmutagenesis, such as after CH1 to produce Fab fragments or after thehinge region to produce F(ab′)₂ fragments. Single chain antibodies maybe produced by joining VL and VH with a DNA linker.

As stated previously, the polynucleotides will be expressed in hostsafter the sequences have been operably linked to (i.e., positioned toensure the functioning of) an expression control sequence. Theseexpression vectors are typically replicable in the host organisms eitheras episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors will contain selection markers, e.g.,tetracycline or neomycin, to permit detection of those cells transformedwith the desired DNA sequences.

E. coli is a prokaryotic host useful particularly for cloning thepolynucleotides of the present invention. Other microbial bosts suitablefor use include bacilli, such as Bacillus subtilus, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any of a number of well-known promoters maybe present, such as the lactose promoter system, a tryptophan (trp)promoter system, a beta-lactamase promoter system, or a promoter systemfrom phage lambda. The promoters will typically control expression,optionally with an operator sequence, and have ribosome binding sitesequences and the like, for initiating and completing transcription andtranslation.

Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention.Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting intact immunoglobulins have beendeveloped in the art, and include the CHO cell lines, various COS celllines, Syrian Hamster Ovary cell lines, HeLa cells, preferably myelomacell lines, transformed B-cells, human embryonic kidney cell lines, orhybridomas. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, cytomegalovirus and the like.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment or electroporation may be used forother cellular hosts.

Once expressed, the antibodies can be purified according to standardprocedures, including ammonium sulfate precipitation, ion exchange,affinity, reverse phase, hydrophobic interaction column chromatography,gel electrophoresis, and the like. Substantially pure immunoglobulins ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses. Once purified,partially or to homogeneity as desired, the polypeptides may then beused therapeutically or prophylactically, as directed herein.

The antibodies (including immunologically reactive fragments) areadministered to a subject at risk for or exhibiting Aβ-related symptomsor pathology such as clinical or pre-clinical Alzheimer's disease,Down's syndrome, or clinical or pre-clinical amyloid angiopathy, usingstandard administration techniques, preferably peripherally (i.e. not byadministration into the central nervous system) by intravenous,intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular,intranasal, buccal, sublingual, or suppository administration. Althoughthe antibodies may be administered directly into the ventricular system,spinal fluid, or brain parenchyma, and techniques for addressing theselocations are well known in the art, it is not necessary to utilizethese more difficult procedures. The antibodies of the invention areeffective when administered by the more simple techniques that rely onthe peripheral circulation system. The advantages of the presentinvention include the ability of the antibody to exert its beneficialeffects even though not provided directly to the central nervous systemitself. Indeed, it has been demonstrated that the amount of antibodythat crosses the bloodbrain barrier is ≦0.1% of plasma levels.

The pharmaceutical compositions for administration are designed to beappropriate for the selected mode of administration, andpharmaceutically acceptable excipients such as, buffers, surfactants,preservatives, solubilizing agents, isotonicity agents, stabilizingagents and the like are used as appropriate. Remington's PharmaceuticalSciences, Mack Publishing Co., Easton Pa., latest edition, incorporatedherein by reference, provides a compendium of formulation techniques asare generally known to practitioners.

The concentration of the humanized antibody in formulations may rangefrom as low as about 0.1% to as much as 15 or 20% by weight and will beselected primarily based on fluid volumes, viscosities, and so forth, inaccordance with the particular mode of administration selected. Thus, apharmaceutical composition for injection could be made up to contain in1 mL of phosphate buffered saline from 1 to 100 mg of the humanizedantibody of the present invention. The formulation could be sterilefiltered after making the formulation, or otherwise mademicrobiologically acceptable. A typical composition for intravenousinfusion could have a volume as much as 250 mL of fluid, such as sterileRinger's solution, and 1-100 mg per mL, or more in antibodyconcentration. Therapeutic agents of the invention can be frozen orlyophilized for storage and reconstituted in a suitable sterile carrierprior to use. Lyophilization and reconstitution can lead to varyingdegrees of antibody activity loss (e.g. with conventional immuneglobulins, IgM antibodies tend to have greater activity loss than IgGantibodies). Dosages may have to be adjusted to compensate. The pH ofthe formulation will be selected to balance antibody stability (chemicaland physical) and comfort to the patient when administered. Generally,pH between 4 and 8 is tolerated.

Although the foregoing methods appear the most convenient and mostappropriate for administration of proteins such as humanized antibodies,by suitable adaptation, other techniques for administration, such astransdermal administration and oral administration may be employedprovided proper formulation is designed. In addition, it may bedesirable to employ controlled release formulations using biodegradablefilms and matrices, or osmotic mini-pumps, or delivery systems based ondextran beads, alginate, or collagen. In summary, formulations areavailable for administering the antibodies of the invention and arewell-known in the art and may be chosen from a variety of options.Typical dosage levels can be optimized using standard clinicaltechniques and will be dependent on the mode of administration and thecondition of the patient.

The following examples are intended to illustrate but not to limit theinvention. Because the examples here describe experiments conducted inmurine systems, the use of murine monoclonal antibodies is satisfactory.However, in the treatment methods of the invention intended for humanuse, humanized forms of the antibodies with the immunospecificitycorresponding to that of antibody 3D6 are preferred.

EXAMPLE 1 Synthesis of Humanized Antibody 3D6

Cells and antibodies. Mouse myeloma cell line Sp2/0 was obtained fromATCC (Manassas, Va.) and maintained in DME medium containing 10% FBS(Cat # SH30071.03, HyClone, Logan, Utah) in a 37° C. CO₂ incubator.Mouse 3D6 hybridoma cells were first grown in RPMI-1640 mediumcontaining 10% FBS (HyClone), 10 mM HEPES, 2 mM glutamine, 0.1 mMnon-essential amino acids, 1 mM sodium pyruvate, 25 μg/ml gentamicin,and then expanded in serum-free media (Hybridoma SFM, Cat # 12045-076,Life Technologies, Rockville, Md.) containing 2% low Ig FBS (Cat #30151.03, HyClone) to a 1.5 liter volume in roller bottles. Mousemonoclonal antibody 3D6 (Mu3D6) was purified from the culturesupernatant by affinity chromatography using a protein-G Sepharosecolumn. Biotinylated Mu3D6 was prepared using EZ-LinkSulfo-NHS-LC-LC-Biotin (Cat # 21338ZZ, Pierce, Rockford, Ill.).

Cloning of variable region cDNAs. Total RNA was extracted fromapproximately 10⁷ hybridoma cells using TRIzol reagent (Cat. # 15596-026Life Technologies) and poly(A)⁺ RNA was isolated with the PolyATractmRNA Isolation System (Cat. # Z5310, Promega, Madison, Wis.) accordingto the suppliers' protocols. Double-stranded cDNA was synthesized usingthe SMART™RACE cDNA Amplification Kit (Cat. # K1811-1, Clontech, PaloAlto, Calif.) following the supplier's protocol. The variable regioncDNAs for the light and heavy chains were amplified by polymerase chainreaction (PCR) using 3′ primers that anneal respectively to the mousekappa and gamma chain constant regions, and a 5′ universal primerprovided in the SMART™RACE cDNA Amplification Kit. For VL PCR, the 3′primer has the sequence:5′-TATAGAGCTCAAGCTTGGATGGTGGGAAGATGGATACAGTTGGTGC-3′ [SEQ ID NO: 13]

with residues 17-46 hybridizing to the mouse Ck region. For VH PCR, the3′ primers have the degenerate sequences:                                A       G   T5′-TATAGAGCTCAAGCTTCCAGTGGATAGACCGATGGGGCTGTCGTTTTGGC-3′ [SEQ ID NO:14]                                Twith residues 17-50 hybridizing to mouse gamma chain CH1. The VL and VHcDNAs were subcloned into pCR4Blunt-TOPO vector (Cat. # 45-0031,Invitrogen, Carlsbad, Calif.) for sequence determination. DNA sequencingwas carried out by PCR cycle sequencing reactions with fluorescentdideoxy chain terminators (Applied Biosystems, Foster City, Calif.)according to the manufacturer's instructionas. The sequencing reactionswere analyzed on a Model 377 DNA Sequencer (Applied Biosystems).

Construction of humanized 3D6 (Hu3D6) variable regions. Humanization ofthe mouse antibody V regions was carried out as outlined by Queen etal., (1989), op. cit. The human V region framework used as acceptor forMu3D6 CDRs was chosen based on sequence homology. The computer programsABMOD and ENCAD [Levitt, M., J. Mol. Biol. 168:595-620 (1983)] were usedto construct a molecular model of the variable regions. Amino acids inthe humanized V regions that were predicted to have contact with CDRswere substituted with the corresponding residues of Mu3D6. This was doneat residues 49, 73, and 98 in the heavy chain and at residue 41 in thelight chain. The amino acids in the humanized V region that were foundto be rare in the same V-region subgroup were changed to the consensusamino acids to eliminate potential immunogenicity. This was done atresidues 6 and 91 in the heavy chain.

The light and heavy chain variable region genes were constructed andamplified using eight overlapping synthetic oligonucleotides ranging inlength from approximately 65 to 80 bases [He, X. Y., et al., J. Immunol.160: 029-1035 (1998)]. The oligonucleotides were annealed pairwise andextended with the Klenow fragment of DNA polymerase I, yielding fourdouble-stranded fragments. The resulting fragments were denatured,annealed pairwise, and extended with Klenow, yielding two fragments.These fragments were denatured, annealed pairwise, and extended onceagain, yielding a full-length gene. The resulting product was amplifiedby PCR using the Expand High Fidelity PCR System (Cat. # 1 732 650,Roche Molecular Biochemicals, Indianapolis, Ind.). The PCR-amplifiedfragments were gel-purified and cloned into pCR4Blunt-TOPO vector. Aftersequence confirmation, the VL and VH genes were digested with MIuI andXbaI, gel-purified, and subcloned respectively into vectors forexpression of light and heavy chains to make pVk-Hu3D6 and pVg1-Hu3D6[Co, M. S., et al., J. Immunol. 148:1149-1154 (1992)]. The maturehumanized 3D6 antibody expressed from these plasmids has the light chainof SEQ ID NO:11 and the heavy chain of SEQ ID NO:12.

Stable transfection. Stable transfection into mouse myeloma cell lineSp2/0 was accomplished by electroporation using a Gene Pulser apparatus(BioRad, Hercules, Calif.) at 360 V and 25 μF as described (Co, et al.,1992, op. cit.). Before transfection, pVk-Hu3D6 and pVg1-Hu3D6 plasmidDNAs were linearized using FspI and BstZ171, respectively. Approximately10⁷ Sp2/0 cells were transfected with 20 μg of pVk-Hu3D6 and 40 μg ofpVg1-Hu3D6. The transfected cells were suspended in DME mediumcontaining 10% FBS and plated into several 96-well plates. After 48 hr,selection media (DME medium containing 10% FBS, HT media supplement, 0.3mg/ml xanthine and 1 μg/ml mycophenolic acid) was applied. Approximately10 days after the initiation of the selection, culture supernatants wereassayed for antibody production by ELISA as shown below. High yieldingclones were expanded in DME medium containing 10% FBS and furtheranalyzed for antibody expression. Selected clones were then adapted togrowth in Hybridoma SFM.

Measurement of antibody expression by ELISA. Wells of a 96-well ELISAplate (Nunc-Immuno plate, Cat # 439454, NalgeNunc, Naperville, Ill.)were coated with 100 μl of 1 μg/ml goat anti-human IgG, Fc γ fragmentspecific, polyclonal antibodies (Cat # 109-005-098, JacksonImmunoResearch, West Grove, Pa.) in 0.2 M sodium carbonate-bicarbonatebuffer (pH 9.4) overnight at 4° C. After washing with Washing Buffer(PBS containing 0.1% Tween 20), wells were blocked with 400 μl ofSuperblock Blocking Buffer (Cat # 37535, Pierce) for 30 min and thenwashed with Washing Buffer. Samples containing Hu3D6 were appropriatelydiluted in ELISA Buffer (PBS containing 1% BSA and 0.1% Tween 20) andapplied to ELISA plates (100 μl per well). As a standard, humanizedanti-CD33 IgG1 monoclonal antibody HuM195 (Co, et al., 1992, op. cit.)was used. The ELISA plate was incubated for 2 hr at room temperature andthe wells were washed with Washing Buffer. Then, 100 μl of1/1,000-diluted HRP-conjugated goat anti-human kappa polyclonalantibodies (Cat # 1050-05, Southern Biotechnology, Birmingham, Ala.) inELISA Buffer was applied to each well. After incubating for 1 hr at roomtemperature and washing with Washing Buffer, 100 μl of ABTS substrate(Cat #s 507602 and 506502, Kirkegaard and Perry Laboratories,Gaithersburg, Md.) was added to each well. Color development was stoppedby adding 100 μl of 2% oxalic acid per well. Absorbance was read at 415nm using an OPTImax microplate reader (Molecular Devices, Menlo Park,Calif.).

Purification of Hu3D6. One of the high Hu3D6-expressing Sp2/0 stabletransfectants (clone #40) was adapted to growth in Hybridoma SFM andexpanded to 2 liters in roller bottles. Spent culture supernatant washarvested when cell viability reached 10% or below and loaded onto aprotein-A Sepharose column. The column was washed with PBS before theantibody was eluted with 0.1 M glycine-HCl (pH 2.5), 0.1 M NaCl. Theeluted protein was dialyzed against 3 changes of 2 liters of PBS andfiltered through a 0.2 μm filter prior to storage at 4° C. Antibodyconcentration was determined by measuring absorbance at 280 nm (1mg/ml=1.4 A₂₈₀). SDS-PAGE in Tris-glycine buffer was performed accordingto standard procedures on a 4-20% gradient gel (Cat # EC6025, Novex, SanDiego, Calif.). Purified humanized 3D6 antibody is reduced and run on anSDS-PAGE gel. The whole antibody shows two bands of approximatemolecular weights 25 kDa and 50 kDa. These results are consistent withthe molecular weights of the light chain and heavy chain, or with themolecular weight of the chain(s) comprising a fragment, calculated fromtheir amino acid compositions.

EXAMPLE 2 It Vitro Binding Properties of Humanized 3D6 Antibody

The binding efficacy of humanized 3D6 antibody, synthesized and purifiedas described above, was compared with the mouse 3D6 antibody usingbiotinylated mouse 3D6 antibody in a comparative ELISA. Wells of a96-well ELISA plate (Nunc-Immuno plate, Cat # 439454, NalgeNunc) werecoated with 100 μl of β-amyloid peptide (1-42) in 0.2 M sodiumcarbonate/bicarbonate buffer (pH 9.4) (0.3 μg/mL) overnight at 4° C.

After washing the wells with phosphate buffered saline (PBS) containing0.1% Tween 20 (Washing Buffer) using an ELISA plate washer, the wellswere blocked by adding 300 μL of SuperBlock reagent (Pierce) per well.After 30 minutes of blocking, the wells were washed with Washing Bufferand excess liquid was removed.

A mixture of biotinylated Mu3D6 (0.2 μg/ml final concentration) andcompetitor antibody (Mu3D6 or Hu3D6; starting at 300 μg/ml finalconcentration and serial 3-fold dilutions) in ELISA Buffer were added intriplicate in a final volume of 100 μl per well. As a no-competitorcontrol, 100 μl of 0.2 μg/ml biotinylated Mu3D6 was added. As abackground control, 100 μl of ELISA Buffer was added. The ELISA platewas incubated at room temperature for 90 min. After washing the wellswith Washing Buffer, 100 μl of 1 μg/ml HRP-conjugated streptavidin (Cat# 21124, Pierce) was added to each well. The plate was incubated at roomtemperature for 30 min and washed with Washing Buffer. For colordevelopment, 100 μl/well of ABTS Peroxidase Substrate (Kirkegaard &Perry Laboratories) was added. Color development was stopped by adding100 μl/well of 2% oxalic acid. Absorbance was read at 415 nm. Theabsorbances were plotted against the log of the competitorconcentration, curves were fit to the data points (using Prism) and theIC50 was determined for each antibody using methods well-known in theart.

The mean IC50 for mouse 3D6 was 2.7 μg/mL (three separate experiments,standard deviation=0.6 μg/mL) and for humanized 3D6 was 3.3 μg/mL (threeseparate experiments, standard deviation=0.8 μg/mL). A second set ofthree experiments was carried out, essentially as described above, andthe mean IC50 for mouse 3D6 was determined to be 3.97 μg/mL (SD=0.15μg/mL) and for humanized 3D6, the IC50 was determined to be 3.97 μg/mL(SD=0.20 μg/mL). On the basis of these results, we conclude thathumanized 3D6 has binding properties that are very similar to those ofthe mouse antibody 3D6. Therefore, we expect that humanized 3D6 has verysimilar in vitro and in vivo activities compared with mouse 3D6 and willexhibit in humans the same effects demonstrated with mouse 3D6 in mice.

EXAMPLE 3 In Vitro Binding Properties of Mouse and Humanized Antibodies3D6

Antibody affinity (KD=Kd/Ka) was determined using a BIAcore biosensor2000 and data analyzed with BIAevaluation (v. 3.1) software. A captureantibody (rabbit anti-mouse or anti-human IgG) was coupled via freeamine groups to carboxyl groups on flow cell 2 of a biosensor chip (CM5)using N-ethyl-N-dimethylaminopropyl carbodiimide andN-hydroxysuccinimide (EDC/NHS). A non-specific rabbit IgG was coupled toflow cell 1 as a background control. Monoclonal antibodies were capturedto yield 300 resonance units (RU). Amyloid-beta 1-40 or 1-42 (BiosourceInternational, Inc.) was then flowed over the chip at decreasingconcentrations (1000 to 0.1 times KD). To regenerate the chip, boundanti-Aβ antibody was eluted from the chip using a wash with glycine-HCl(pH 2). A control injection containing no amyloid-beta served as acontrol for baseline subtraction. Sensorgrams demonstrating associationand dissociation phases were analyzed to determine Kd and Ka. Theaffinity (KD) of mouse antibody 3D6 for Aβ 1-42 was determined to be 2.4nM, and the affinity of humanized 3D6, prepared essentially as describedin Example 1, was determined to be 2.3 nM.

EXAMPLE 4 Epitope Mapping of Mouse and Humanized 3D6

The BIAcore is an automated biosensor system for measuring molecularinteractions [Karlsson R., et al. J Immunol. Methods 145:229-240(1991)]. The advantage of the BIAcore over other binding assays is thatbinding of the antigen can be measured without having to label orimmobilize the antigen (i.e. the antigen maintains a more nativeconformation). The BIAcore methodology was used to assess the binding ofvarious amyloid-beta peptide fragments to either mouse 3D6 or humanized3D6 (prepared substantially as described in Example 1). All dilutionswere made with HEPES buffered saline containing Tween 20. A singleconcentration of a variety of fragments of human Aβ or mouse Aβ 1-40(BioSource International) was used. Human amyloid beta fragments 1-10and 1-20 bound to mouse 3D6 and to humanized 3D6, while human Aβfragments 10-20 and 16-25 did not bind to either antibody. Neither mouse3D6 nor humanized 3D6 bound mouse Aβ1-40. Using this methodology, thebinding epitope for both mouse and humanized 3D6 appears to be betweenamino acids 1 and 10 of human Aβ.

EXAMPLE 5 Effects of Administration of 3D6

Unless otherwise stated, all studies used APP^(V717F) (PDAPP) transgenicmice, and all injections were i.p. In general, a control group of micereceived injections of saline.

Six weeks of weekly injection of 360 μg of 3D6 in old, hemizygous mice(24 month) lowered hippocampal insoluble Aβ_(total) by 10% and Aβ1-42 by1% (N.S., not statistically significant) in 9 animals per control groupand 10 animals per antibody group. In the cortex, mean insolubleAβ_(total) was lower by 33% and Aβ 1-42 by 47% (p<0.05), while insolubleAβ 1-40 increased by 100%.

In hemizygous, 4 month old mice, administration of 360 μg of 3D6 peranimal: 1) raised average plasma Aβ1-40 and Aβ1-42 levels approximately6-fold and 9-fold, respectively, by 24 hours after administration; and2) had no significant effect on soluble Aβ 1-40 in the cortex after 24hours compared with saline control (5 animals per group). In anotherstudy with hemizygous, 3 month old mice, administration of 360 μg of 3D6per animal raised average plasma Aβ1-42 levels approximately 8-fold by24 hours after administration.

Administration of 360 μg of 3D6 per animal (5 animals per group, salinecontrol): raised average plasma Aβ 1-40 and Aβ 1-42 levels approximately92-fold and 32-fold, respectively, by 24 hours after administration(p<0.05); lowered cortical insoluble Aβ 1-40 by 42% (p<0.05) and Aβ 1-42by 27% (N.S.), but increased Aβ_(total) by 35% (N.S.); had no consistentor significant effect on soluble or insoluble Aβ 1-40, Aβ 1-42, orAβ_(total) in the hippocampus after 24 hours; in the cerebellum,increased soluble Aβ 1-42 by 80% (p<0.001) and Aβ_(total) by 68% (N.S.),but lowered soluble Aβ 1-40 by 0.6% (N.S.); and in the cerebellum,lowered insoluble Aβ 1-40, Aβ 1-42, and Aβ_(total) by 35% (p<0.01), 21%(N.S.), and 12% (N.S.), respectively.

In young mice, administration of 360 μg of 3D6 per animal (5 pergroup): 1) raised average plasma Aβ 1-42 levels approximately 3-fold by24 hours after administration; and 2) in the cortex, lowered insolubleAβ 1-40 about 10% and increased insoluble Aβ 1-42 about 12%.

Studies were conducted to assess the effects of 3D6 on formation ofstable Aβ:antibody complexes in biological fluids, plasma Aβconcentrations acutely after administration, cognitive performance afteracute or chronic administration, and guanidine-extracted andimmunohistochemically-detected Aβ deposition (in brain) after chronicadministration.

Mice (3 months of age) were injected with 360 μg of 3D6. Twenty-fourhours following antibody administration plasma was collected andproteins were resolved by gel electrophoresis under native(non-denaturing conditions) on a polyacrylamide gel. Following transferof size fractionated proteins to a solid matrix, complexes wereimmunodetected with biotinylated antibody and visualized with enhancedchemiluminescence. Unlike certain other anti-Aβ antibodies, no complexwas detected with 3D6.

Young (2-3 months of age) mice were injected with 3D6. At various timesfollowing antibody administration, plasma was collected and various Aβspecies were determined by a sandwich ELISA. Administration of 3D6resulted in a dose-and time-dependent increase in plasma Aβ levels.Aβ₁₋₄₀ levels increased to a greater degree than Aβ₁₋₄₂ levels following3D6 administration. In an additional study, young APP^(V717F) tg micewere treated with 360 μg 3D6 and plasma AD levels were measured at 0.5,3, 6, and 24 h following injection. 3D6 increased plasma Aβ levels in atime-dependent manner.

Extensive behavioral characterization of APP^(V717F)tg mice has beenperformed using several memory paradigms (bar-press, 8 arm-radial maze,object recognition). These mice are impaired in several learning andmemory tasks, and deficits in the object recognition (OR) task worsenwith age. Therefore, the OR task has been used to assess learning andmemory in APP^(V717F)tg mice. Performance in the OR task ispreferentially dependent on the integrity of the medial temporal lobe(perirhinal and entorhinal cortices). The OR test relies on thespontaneous tendency of rodents to preferentially explore a novel versusfamiliar object.

On the first day of testing, mice were allowed to habituate to an openfield chamber for 50 minutes. The following day, mice were placed backinto the open field for two 10-min trials. During trial one, mice wereallowed to explore the open field in the presence of an object (e.g.,marble or die). Following a 3-hr inter-trial delay, mice were placedback into the open field with the familiar object (the same objectexplored previously during trial 1) as well as a novel object. The timespent exploring the novel object as well as the familiar object wasrecorded and a recognition index (the ratio of time spent exploring thenovel object×100/total time spent exploring both objects) was calculatedfor each mouse. Administration of 360 μg of 3D6 per animal 24 hoursprior to the habituation session in 11-12 month old APP^(V717F)tg miceimproved OR performance in 2 of 8 mice tested (p<0.05).

Homozygous tg mice (5-6 months old) were administered weekly injectionsof PBS and 72, 217, and 360 μg of a non-specific IgG or 3D6 (n=19-30)for 5 months. At necropsy, the brains were removed and processed for AβELISA assays and immunohistochemical analysis of parenchymal Aβ burden.Cortical and hippocampal tissues were homogenized in PBS. PBS-insolubleAβ was subsequently extracted from the pellets by homogenization in 5.5M guanidine-HCl. Following homogenization, the samples were nutated forat least 24 h prior to centrifugation and collection of the guanidineextract. PBS-soluble and guanidine-extracted tissue preparations werestored at −80° C. for subsequent Aβ ELISA determinations.Immunohistochemical (IHC) analysis of parenchymal Aβ burden was carriedout as follows. Eight (8) μm paraffin embedded paraformaldehyde fixedtissues were labeled with rabbit polyclonal anti-Aβ antibody (against Aβ15-30) and followed by anti-rabbit IgG fluorescent detection. Eight (8)sections of brain (7 IHC, 1 control) were examined from each animal.Treatment with 3D6 (360 μg) markedly and significantly reduced corticalguanidine-extracted Aβ1-42 (by ELISA) and cortical and hippocampal Aβplaque burden (by IHC), but no effect was observed at lower 3D6 doses.Although no effect on guanidine extracted Aβ1-42 was observed at lower3D6 doses, these doses significantly reduced cortical and hippocampal Aβplaque burden (by IHC).

Radiolabeled (15 μCi/mouse, 0.5 mg/mouse) 3D6 was administered to ICR(non-transgenic) mice in order to evaluate kinetics and braindistribution of the antibody after administration by the intravenousroute. Plasma kinetics for 3D6 immunoreactivity demonstrated a half-lifeof elimination of approximately 5 days. TCA-precipitable radioactivitywas greater than 95% of the total plasma counts throughout the study,and declined in the plasma compartment with a terminal half-life of 3-4days. The observation that plasma radioactivity remained predominantlyTCA-precipitable throughout the study suggests that the radiolabeledantibody was not significantly proteolytically degraded, nor was the125-I label cleaved from the antibody over the time course studied. Theshapes of the concentration versus time profiles as measured by ELISAand radioactivity were generally similar, with some differences in theterminal phases. There was no apparent accumulation of radiolabel in anytissue, including brain. Distribution of radioactivity to the brain wasminimal. The amount of radioactivity associated with the brain samplesin this experiment cannot be clearly distinguished from contamination bythe blood compartment during tissue processing or from antibodyassociated with endothelial cells in the brain vasculature.

Nine month old, hemizygous mice received PBS, a non-specific IgG, or 3D6(500 μg/week) by weekly injection for six months (PBS, 11 animals; IgG,13 animials; and 3D6, 14 animals). Weak, but statistically significant,Aβ lowering in the cortex (compared to IgG) and hippocampus (compared toIgG or combined PBS/IgG controls) was seen. Immunohistochemical (IHC)analysis showed strong reductions in Aβ plaque burden in the cortex andhippocampus of 3D6-treated mice (94% and 85% reductions, respectively,versus PBS control; p<0.05, and p<0.01, respectively).

EXAMPLE 6 Administration of Humanized 3D6

A preparation of an anti-Aβ antibody comprising a light chain having theamino acid sequence of SEQ ID NO:11 and a heavy chain having the aminoacid sequence of SEQ ID NO:12 (a humanized 3D6) was administered as asingle intravenous bolus injection to two groups of 12 male marmosets atdoses of 1 and 10 mg/kg. Concentrations of immunoreactive anti-Aβantibody declined with a half-life of elimination of approximately 4days. C_(max) and AUC parameters increased proportionally between the 1and 10 mg/kg dose levels. The administration of humanized 3D6 tomarmosets resulted in 18 or 29-fold increase in plasma Aβ₁₋₄₀immunoreactivity after 8 hours, compared with predose concentrations inthe 1 and 10 mg/kg dose groups, respectively. Animals at both doselevels had concentrations of Aβ₁₋₄₀ immunoreactivity above baselinelevels up to 2 weeks after antibody administration. Kinetic analysis ofconcentrations of Aβ₁₋₄₀ immunoreactivity showed that the half-life ofelimination of Aβ₁₋₄₀ immunoreactivity was comparable to that of theantibody (˜4 days). The pharmacokinetics of humanized 3D6 were alsoevaluated in male cynomolgus monkeys after a single intravenousadministration of 1 mg/kg. Analysis of immunoreactivity showed thathumanized 3D6 was eliminated from the plasma with a half-life ofapproximately 11-12 days.

1-29. (canceled)
 30. An antibody comprising the sequence given by SEQ IDNO:9 and the sequence given by SEQ ID NO:10.
 31. A polynucleotidecompound, comprising a sequence coding for either SEQ ID NO:9 or SEQ IDNO:10 of the antibody of claim
 30. 32. A cell that is capable ofexpressing the antibody of claim
 30. 33. A pharmaceutical composition,comprising the antibody of claim 30 and a pharmaceutically acceptableexcipient.
 34. A method for treating clinical or pre-clinicalAlzheimer's disease, Down's syndrome, or clinical or pre-clinicalamyloid angiopathy in a human subject, comprising administering to thehuman subject an effective amount of the antibody of claim
 30. 35. Anantibody comprising a light chain comprising the sequence given by SEQID NO:9 and a heavy chain comprising the sequence given by SEQ ID NO:10.36. A polynucleotide compound, comprising a sequence coding for eitherSEQ ID NO:9 or SEQ ID NO:10 of the antibody of claim
 35. 37. A cell thatis capable of expressing the antibody of claim
 35. 38. A pharmaceuticalcomposition, comprising the antibody of claim 35 and a pharmaceuticallyacceptable excipient.
 39. A method for treating clinical or pre-clinicalAlzheimer's disease, Down's syndrome, or clinical or pre-clinicalamyloid angiopathy in a human subject, comprising administering to thehuman subject an effective amount of the antibody of claim
 35. 40. Theantibody of claim 35 having a light chain of the sequence given by SEQID NO:11 and a heavy chain of the sequence given by SEQ ID NO:12.
 41. Apolynucleotide compound, comprising a sequence coding for either SEQ IDNO:11 or SEQ ID NO:12 of the antibody of claim
 40. 42. A cell that iscapable of expressing the antibody of claim
 40. 43. A pharmaceuticalcomposition, comprising the antibody of claim 40 and a pharmaceuticallyacceptable excipient.
 44. A method for treating clinical or pre-clinicalAlzheimer's disease, Down's syndrome, or clinical or pre-clinicalamyloid angiopathy in a human subject, comprising administering to thehuman subject an effective amount of the antibody of claim
 40. 45. Anantibody fragment comprising the sequence given by SEQ ID NO:9 and thesequence given by SEQ ID NO:10.
 46. The antibody fragment of claim 45,which is an Fab or an F(ab′)2 fragment.
 47. The antibody fragment ofclaim 45, which is a single chain.
 48. A polynucleotide compound,comprising a sequence coding for either SEQ ID NO:9 or SEQ ID NO:10 ofthe antibody fragment of claim
 45. 49. A cell that is capable ofexpressing the antibody fragment of claim
 45. 50. A pharmaceuticalcomposition, comprising the antibody fragment of claim 45 and apharmaceutically acceptable excipient.
 51. A method for treatingclinical or pre-clinical Alzheimer's disease, Down's syndrome, orclinical or pre-clinical amyloid angiopathy in a human subject,comprising administering to the human subject an effective amount of theantibody fragment of claim 45.